Embryonic stem cells are thought to have a significant potential for regenerative medicine. The use of nuclear transfer for the generation of customized ES cells offers the possibility for patient-specific therapy. However, embryonic stem cell and nuclear transfer technology are highly controversial because of unresolved technical issues with human cloning and because of serious ethical objections to the use of human embryos. The key issue of the stem cell field is to accomplish reprogramming of a somatic into a pluripotent cell in the culture dish without the use of eggs. New results from our and from other laboratories demonstrate that reprogramming to pluripotency can be achieved without exposure of the somatic nucleus to the egg cytoplasm, by retrovirus-mediated transfer of Oct4, Sox2, c-myc and Klf4 into fibroblasts. The reprogrammed cells were shown to be molecularly and biologically indistinguishable from normal ES cells. This proposal has 4 aims: 1. We will define the molecular mechanism of reprogramming. 2. We will establish high throughput screens to identify small molecules that increase efficiency of reprogramming and substitute for the need to introduce any of the factors by retrovirus-mediated gene transfer. 3. Because only a small fraction of cells are converted to a pluripotent state we will define the target cells of reprogramming. Also, we will assess whether other cell types than fibroblasts can be reprogrammed. 4. The present protocols use transgenic donor cells for the selection of pluripotent cells in culture, which constitutes a major obstacle if the approach is ever to be contemplated for transplantation medicine. We will seek to establish alternative screening methods that could be used to derive reprogrammed cells from fibroblasts of non-transgenic mice. Finally, we will establish proof of principle of the therapeutic potential of reprogrammed cells in a model for Parkinson's and Sickle Cell Disease. The long-term goal of this project is to understand the molecular events that accomplish changing the epigenetic state of a somatic cell to a pluripotent state so the process can eventually be used for the treatment of patients with degenerative disease. In vitro Reprogramming of Somatic Cells into Pluripotent ES-Like Cells Public Health Relevance: The promise of embryonic stem cells in combination with nuclear transplantation is to provide patient-specific cell therapy for many degenerative diseases, but any clinical application of this approach remains highly controversial due to scientific problems and ethical objections. A potential solution to these problems is in vitro reprogramming of somatic cells to pluripotent ES cells that could be used for customized therapy. This proposal is based on the successful in vitro generation of ES cells from fibroblasts and seeks to solve some of the key problems that need to be worked out before the approach can be considered for the potential application to medicine.